The cholinergic parasympathetic input to the salivary glands of cat, rat, and presumably mouse contain both acetylcholine (ACh) and vasoactive intestinal peptide (VIP). We wished to use this gland as a model for studying the functional interrelationships of such a coexistence of neurotransmitters. Although it is known that cholinergic drugs regulate release of both VIP and ACh in salivary gland, it was not known what the effects would be of VIP on the cholinergic input in vivo. In order to examine this question we developed a new technique for measurement of acetylcholine content, and used this method to measure turnover in mice infused with VIP, or injected with a muscarinic agonist (pilocarpine) or antagonist (atropine). Pilocarpine and VIP both decreased ACh turnover in mouse salivary gland; atropine increased turnover rate. The data suggest that there is a feedback loop, possibly neuronal in nature that regulates cholinergic activity. By changing postsynaptic receptor function, VIP apparently participates in the feedback regulation of ACh metabolism.